IBC-1 (invasive breast cancer-1), a putative oncogene amplified in breast cancer

ABSTRACT

This invention encompasses antibodies specific for IBC-1 (Invasive Breast Cancer-1), methods for diagnosis and prognosis of metastatic breast cancer and degenerative neural conditions, methods of identifying and manufacturing therapeutic compounds, and methods of treating patients with invasive and metastatic breast cancer or degenerative neural conditions.

RELATED APPLICATIONS

This application is a continuation-in-part, and claims priority, ofInternational Application No. PCT/US02/34499, filed Oct. 28, 2002, whichclaims priority of U.S. Provisional Application Ser. No. 60/343,154,filed Oct. 26, 2001, and U.S. Provisional Application Ser. No.60/356,301, filed Feb. 12, 2002, the contents of all of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

This invention relates to cancer and neurodegenerative diseases.

BACKGROUND

Breast cancer is a leading cause of cancer deaths in women worldwide.Despite recent improvements in cancer therapy, advanced stage tumors arestill almost inevitably fatal (Alberg et al. (2000) Curr Opin Oncol12:515-520). Therefore, there is a need for the identification of noveltherapeutic targets, particularly in estrogen receptor negative andmetastatic tumors which are the least responsive to current therapies.

SUMMARY

This invention is based on identification of a human gene that isexpressed in an aggressive subset of invasive breast carcinomas and inthe pons of the brain, but not in 75 other normal human adult and fetaltissues. This gene was designated IBC-1 (Invasive Breast Cancer-1). Thepredicted amino acid sequence encoded by IBC-1 cDNA contains sequencesimilar or identical to that of (a) a previously identified humancachexia-associated protein (Akerblom et al., U.S. Pat. No. 5,834,192),(b) a protein fragment derived from PIF (Proteolysis Inducing Factor;also called cancer cachexia factor), (c) a putative secreted neuralsurvival peptide (Cunningham et al. (1998) J Neurosci 18:7047-7060); andTodorov et al. (1996) Nature 379:739-742), and (d) a dermcidin proteinexpressed in sweat glands of the skin (Schittek et al. (2001) NatureImmunology 2:1133-1137). IBC-1 encodes a 110 amino acid pro-protein(i.e., “pro-IBC-1;” SEQ ID NO:3) with a predicted 19 amino acid signalpeptide at the N-terminal, which is presumably removed upon maturationof the protein.

As used herein, “pro-IBC-1” refers to the 110 amino acid protein with aputative signal peptide, whereas “IBC-1” refers to the 91 amino acidmature protein (i.e., amino acids 20-110 of pro-IBC-1; SEQ ID NO:4).

This invention relates to antibodies specifically binding to IBC-1,methods of diagnosing and prognosticating cancer and neural diseases,methods of identifying and manufacturing a therapeutic compound, andmethods of treating cancer and neural diseases. The cancer can be, forexample, a breast cancer, pancreatic cancer, brain cancer, gastriccancer, lung cancer, lymphoma, or any other type of cancer that isinvasive and metastatic and that may or may not be associated withcachexia.

More specifically, this invention includes a purified antibody thatspecifically binds to an epitope within a fragment of IBC-1, e.g.,within (or in) a fragment that includes or consists of the sequence of:SEQ ID NO:5 (amino acids 20-42 of pro-IBC-1); SEQ ID NO:6 (amino acids43-64 of pro-IBC-1); SEQ ID NO:7 (amino acids 53-64 of pro-IBC-1); SEQID NO:12 (amino acids 45-64 of pro-IBC-1); or SEQ ID NO:13 (amino acids86-103 of pro-IBC-1). The antibody can be a monoclonal antibody or apolyclonal antibody. These antibodies can be used for detecting IBC-1 ina test sample from a patient, identifying a therapeutic compound, andtreating diseases associated with overexpression of IBC-1.

As used herein, the term “antibody” refers not only to whole antibodymolecules, but also to antigen-binding fragments, e.g., Fab, F(ab′)₂,Fv, and single chain Fv (ScFv) fragments. Also included are chimericantibodies, such as humanized antibodies.

The term “purified antibody,” as used herein, refers to an antibodywhich either has no naturally-occurring counterpart or has beenseparated from components which naturally accompany it, e.g., bloodcells.

Also within the invention are methods of diagnosis and prognosis. Onesuch method is based on determining whether a test sample contains areceptor for IBC-1. The method involves: (a) providing a test samplefrom a human patient; (b) contacting the test sample with a polypeptidecontaining 10-91 consecutive amino acids of IBC-1; and (c) determiningwhether the polypeptide binds to the test sample. Detection of thepolypeptide bound to the test sample in an amount higher than a negativecontrol indicates that the test sample contains a receptor for IBC-1.The polypeptide can be, for example, IBC-1 itself, or a fragment ofIBC-1, 10 to 91 amino acids in length (e.g., 10 to 50 amino acids,preferably 12 to 40 amino acids, and more preferably 15 to 30 aminoacids in length). The test sample can be prepared from a breast cancertissue sample if the patient is suspected of having, or being likely todevelop, invasive and metastatic breast cancer. In such a case, thepresence of the receptor in the test sample in an amount higher than acontrol sample indicates that the patient has, or is likely to develop,invasive and metastatic breast cancer. If the patient is suspected ofsuffering from, or being at risk for developing, a degenerative neuralcondition, the test sample can be prepared from a brain tissue sample,such as a substantia nigra, pons, or hypothalamus tissue sample. In sucha case, an amount of the receptor in the test sample less than an amountof the receptor in a sample from a normal human (i.e., a person withoutany degenerative neural condition) indicates that the patient issuffering from, or at risk for developing, a neural condition involvingdegeneration of substantia nigra, pons or hypothalamus cells, or otherneural cells normally protected by the presence of IBC-1.

Another method of diagnosis and prognosis is based on determiningwhether a test sample contains the IBC-1 mRNA or IBC-1. The methodinvolves: (a) providing a test sample from a patient; and (b) detectingIBC-1 rMRNA or IBC-1 in the test sample. If the patient is suspected ofhaving, or being likely to develop, invasive and metastatic breastcancer, the test sample can be prepared from a breast cancer tissuesample, or a body fluid (e.g., urine, breast milk, saliva, or blood);the presence of a higher than control level of IBC-1 mRNA or IBC-1 inthe test sample indicates that the patient has, or is likely to develop,invasive and metastatic breast cancer. If the patient is suspected ofsuffering from, or being at risk for developing, a degenerative neuralcondition, the test sample is typically prepared from a substantianigra, pons or hypothalamus tissue sample, or from a body fluid (e.g.,urine, cerebro-spinal fluid, saliva, or blood). An amount of IBC-1 MRNAor IBC-1 in the test sample less than an amount of IBC-1 MRNA or IBC-1in a comparable sample from a normal human indicates that the patient issuffering from, or at risk for developing, a neural condition involvingdegeneration of substantia nigra, pons or hypothalamus cells, or othertypes of neural cells that normally express IBC-1.

A third method of diagnosis and prognosis is based on determiningwhether genomic IBC-1 DNA is amplified in a test sample. The methodinvolves: (a) providing a test sample comprising genomic DNA from abreast cancer patient; and (b) determining whether genomic IBC-1 DNA isamplified in the test sample. If the patient is suspected of having, orbeing likely to develop, invasive and metastatic breast cancer, the testsample can be prepared from a breast cancer tissue sample; the presenceof amplified genomic IBC-1 DNA in the test sample indicates that thepatient has, or is likely to develop, invasive and metastatic breastcancer. As used herein, “amplified” means that the amount of genomic DNAsequences in a cell that can be transcribed into mRNA molecules thatencode functional IBC-1 protein molecules is higher than that in acontrol person (e.g., a person without breast cancer).

Also included in this invention is a method for identifying a compoundthat blocks binding of IBC-1 to its receptor. The method involves: (a)providing a polypeptide that contains between 10 and 91 consecutiveamino acids of IBC-1 and binds an IBC-1 receptor; (b) providing a cellexpressing the IBC-1 receptor; (c) contacting the cell with thepolypeptide in the presence of a test compound; and (d) determiningwhether the test compound blocks binding of the polypeptide to the cell,as an indication that the compound blocks binding of IBC-1 to itsreceptor. The polypeptide can be, for example, IBC-1 itself, or afragment thereof at least 10 amino acids in length (preferably at least12 amino acids, more preferably at least 15 amino acids, e.g., at least20 or at least 50 amino acids in length). Useful IBC-1 fragments can beany IBC-1 fragments disclosed herein. The cell can be a breast cancercell (e.g., an invasive breast cancer cell) or a neural cell (e.g., asubstantia nigra, pons or hypothalamus cell), or any other cell thatexpresses the receptor. The test compound can be, for example, apeptide, a non-peptide small molecule, or an antibody that binds toIBC-1 or its receptor. A compound thus identified can be used fortreating diseases associated with overexpression of IBC-1. If it blocksby binding to IBC-1, it can also be used for detecting IBC-1 in a sample(e.g., for diagnosis and prognosis as described above). If it blocks bybinding to the IBC-1 receptor, it can also be used to detect thepresence of the receptor on a cell (e.g., for diagnosis and prognosis asdescribed above). Once a compound that blocks binding of IBC-1 to itsreceptor has been identified, it can be manufactured in a large scale.

In another aspect, this invention provides a method of treating cancer.The method involves: (a) identifying a patient having, or being likelyto develop, an invasive and metastatic breast cancer that expressesIBC-1 or an IBC-1 receptor; and (b) treating the patient with (i) acompound that blocks binding of IBC-1 to its receptor (e.g., anon-agonistic antibody that binds to IBC-1 or its receptor) or (ii) acompound that inhibits expression of IBC-1 or its receptor (e.g., anRNAi molecule). The patient may or may not be diagnosed as sufferingfrom cachexia, or exhibiting overt symptoms of cachexia (e.g.,unintentional loss of at least 10% of body weight in a short period oftime).

Yet another aspect of this invention is a method of treating a neuralcondition by the steps of (a) identifying a patient suffering from, orat risk for developing, a neural condition involving degeneration ofsubstantia nigra, pons or hypothalamus cells, or another type of neuralcell that normally expresses IBC-1; and (b) administering IBC-1 or anIBC-1 agonist to the patient.

Also within the scope of the invention is a kit for detecting invasiveand metastatic breast cancer. The kit comprises (a) an agent fordetermining the level of IBC-1 in a biological sample, or (b) an agentfor determining whether genomic IBC-1 DNA is amplified in a biologicalsample; and instructions for use of the agent for detecting invasive andmetastatic breast cancer.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention. All publications, patentapplications, patents and other references mentioned herein areincorporated by reference in their entirety. The materials, methods, andexamples disclosed herein are illustrative only and not intended to belimiting. Other features, objects, and advantages of the invention willbe apparent from the description and the accompanying drawings, and fromthe claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a diagram of the genomic structure of the IBC-1 gene.Exon-intron boundaries, start and stop codons, and the SAGE tag and theadjacent NlaIII site (SEQ ID NO:8) that led to the identification ofIBC-1 are indicated.

FIG. 1B is a representation of the nucleic acid sequence of a cDNA (SEQID NO:1) encoding pro-IBC-1 and the predicted amino acid sequence (SEQID NO:3) of pro-IBC-1. Sequences of peptides derived from a cachecticfactor and a neural survival peptide are indicated by thick and thinunderlines, respectively. A predicted secretory signal peptidasecleavage site is marked by an arrow.

FIG. 1C is an amino acid sequence alignment of IBC-1 (i.e., pro-IBC-1;SEQ ID NO:3), lacritin (SEQ ID NO:10), and EST-AI12471 (SEQ ID NO:11)proteins. Amino acids identical to the consensus are shaded. Comparisonwas made using DNAStar and the Clustal algorithm.

FIG. 2A is a schematic representation of control alkaline-phosphatase(AP) protein, alkaline-phosphatase-IBC-1 fusion (AP-IBC-1) protein, andbinding of AP-IBC-1 ligand to a hypothetical membrane protein.

FIG. 2B is a bar graph depicting relative levels of AP activity bound tovarious human cell lines in the form of AP-IBC-1 protein. Cell lines arelisted on the x-axis, while the y-axis indicates bound alkalinephosphatase activity expressed as OD/hour/ml. “G361+HID-5” and“G361+IBC-1” on the x-axis indicate that G361 cells were pre-incubatedwith purified recombinant HID-5/psoriasin (1.93 mM) and IBC-1 (0.567mM), respectively, prior to AP-IBC-1 (25 nM) binding. Decreased AP-IBC-1binding in the presence of IBC-1, but not in the presence ofHID-5/psoriasin, indicates that the binding is specific for IBC-1.

FIG. 2C is a line graph illustrating binding of AP-IBC-1 to G361 cellsover various concentrations of AP-IBC-1. The insert shows Scatchardtransformation.

DETAILED DESCRIPTION

This invention is based on the identification and characterization of agene encoding IBC-1. IBC-1 was identified as a SAGE tag with no match inthe Unigene and Genbank databases. It was represented only in those SAGElibraries that were generated from invasive and metastatic breastcarcinomas. Subsequent analyses revealed that the predicted amino acidsequence of the IBC-1 protein contains sequences identical to apreviously identified human cachexia-associated protein, a trypticpeptide derived from a previously described proteolysis/cachexiainducing factor (PIF), a neuronal survival peptide, and a dermcidinprotein.

In the panel of breast tumors studied, all tumors that expressed IBC-1were found to be high-nuclear grade and overexpressing erbB2. Tumorswith these characteristics have a poor clinical prognosis, and are lesscommon in postmenopausal women. Importantly, primary tumors in whichIBC-1 protein was detected were more likely to be stage 2 or stage 3breast cancers than stage 1 breast cancer. Tumor stage is determinedaccording to the summary of 3 scores that are given based on tumors size(T), lymph nodes (N), and distant metastasis (M). Stage 1 tumors areTIN0M0 tumors. Stage 2 tumors are either large invasive tumors withoutlymph nodes and distant metastases (T2N0M0) or small invasive tumorswith lymph nodes (TIN1M0). Stage 3 tumors are primary invasive tumorswith lymph nodes but no distant metastases (T2-3N1-2M0). Therefore,expression of IBC-1 defines a clinically relevant sub-group of tumorsand represents a new therapeutic target for the treatment of thesetumors as well as cancer-associated cachexia.

In addition to a subset of invasive breast carcinomas, IBC-1 is alsoexpressed in the pons and paracentral gyrus of the brain, but not in anyother normal adult or fetal tissues tested. The restricted expressionpattern makes IBC-1 a good candidate cancer diagnostic marker andtherapeutic target. The secreted nature and extracellular mechanism ofIBC-1 action make it even more attractive for such potential uses.Consistent with this, the inventors demonstrated the existence of a cellsurface IBC-1 binding protein (i.e., putative IBC-1 receptor) in breastcancer and neuronal cells in vivo. In addition, tumors that expressIBC-1 appear to have more, or higher affinity, IBC-1 binding proteins onthe cell surface, which can further facilitate the potential therapeutictargeting of the IBC-1 pathway in these cells.

Previous studies showed that a 30 amino acid peptide corresponding to aportion of the predicted IBC-1 amino acid sequence appears to protectcells from oxidative insult-induced apoptosis (Cunningham et al. (1998)J Neurosci 18:7047-7060). Neurons are particularly sensitive to reactiveoxygen species (ROS), whereas tumor cells themselves produce largeamounts of ROS (Szatrowski and Nathan (1991) Cancer Res 51:794-798).Therefore, the high expression of IBC-1 in these cell types is likely tobe essential for their survival.

Interestingly, all the neurons that strongly bind IBC-1 in the brainplay a direct or indirect role in the regulation of energy homeostasis.Noradrenergic neurons of the locus ceruleus have projections to allmajor parts of the brain and spinal cord, and are involved inmaintaining vigilance (arousal) status. Similarly, dopaminergic neuronsof the substantia nigra are connected to the cortex, spinal cord, andhypothalamus, and regulate initiative behavioral responses. Finally, andmost interestingly, the Jateral hypothalamus is thought to be a “feedingcenter” and damaging its neurons leads to impaired food intake (Inui(1999) Cancer Res 59:4493-4501). Strong cell surface binding of IBC-1 tothese cells supports a role for IBC-1 in regulating feeding behavior.

In addition, catecholaminergic (noradrenergic and dopaminergic) neuronsare particularly susceptible to oxidative stress, since the biosynthesisof these neurotransmitters from tyrosine requires molecular oxygen.Moreover, the auto-oxidization of catecholamines, the end product ofwhich is melanin that accumulates in neurons of the substantia nigra andlocus ceruleus, leads to the generation of ROS (H₂O₂, O₂ ⁻, and OH⁻).The strong binding of IBC-1 to these neurons is consistent with itsputative role as a neural survival factor that protects againstoxidative stress. In contrast to its low and restricted normalexpression pattern, the aberrant overexpression of IBC-1 by certaincarcinomas leads to elevated circulating IBC-1 protein levels (Wigmoreet al. (2000) Br J Surg 87:53-58; and Cabal-Manzano et al. (2001) Br JCancer 84:1599-1601). Due to its small size, the IBC-1 protein is ableto cross the blood-brain barrier (Cunningham et al. (1998) J Neurosci18:7047-7060). Therefore, elevated systemic IBC-1 levels increase thebinding of IBC-1 to neurons of the pons, midbrain, and hypothalamus,resulting in altered feeding behavior that in combination with increasedmuscle wasting leads to cancer related weight loss. Tumors of breastcancer patients with cachexia were found to be more resistant tochemotherapy than those of patients without significant weight loss(Dewys et al. (1980) Am J Med 69:491-497), consistent with IBC-1 playinga role in the regulation of breast cancer cell survival and feedingbehavior.

IBC-1 Antibodies

This invention features antibodies that bind to an epitope within afragment of the IBC-1 protein, e.g., SEQ ID NO:5(YDPEAASAPGSGNPCHEASAAQK, amino acids 20-42 of pro-IBC-1), SEQ ID NO:6(ENAGEDPGLARQAPKPRKQRSS, amino acids 43-64 of pro-IBC-1), SEQ ID NO:7(RQAPKPRKQRSS, amino acids 53-64 of pro-IBC-1), SEQ ID NO:12(AGEDPGLARQAPKPRKQRSS, amino acids 45-64 of pro-IBC-1), or SEQ ID NO:13(DAVEDLESVGKGAVHDVK, amino acids 86-103 of pro-IBC-1). These fragmentsare predicted to be antigenic and localized on the surface of theprotein by analysis using MacVector, and thus particularly useful ingenerating IBC-1 antibodies.

Such antibodies can be polyclonal antibodies derived from the serum orplasma of animals (e.g., mice, hamsters, gerbils, rabbits, rats, guineapigs, sheep, horses, goats, cows, or pigs) that have been immunized withintact IBC-1 or a portion thereof containing the relevant IBC-1 epitopeusing methods, and optionally adjuvants, known in the art. Suchpolyclonal antibodies can be isolated from serum or plasma by methodsknown in the art.

Monoclonal antibodies that bind to the above IBC-1 fragments are alsoencompassed by the invention. Methods of making and screening monoclonalantibodies are well known in the art.

Once the desired antibody-producing hybridoma has been selected andcloned, the resultant antibody can be produced by a number of methodsknown in the art. For example, the hybridoma can be cultured in vitro ina suitable medium for a suitable length of time, followed by therecovery of the desired antibody from the supernatant. The length ofculture time and medium are known or can be readily determined.

Additionally, recombinant antibodies specific for an IBC-1 fragmentdescribed above, such as chimeric and humanized monoclonal antibodiescomprising both human and non-human portions, are within the scope ofthe invention. Such chimeric and humanized monoclonal antibodies can beproduced by recombinant DNA techniques known in the art, for example,using methods described in Akira et al., European Patent Application184,187; Taniguchi, European Patent Application 171,496; Morrison etal., European Patent Application 173,494; Neuberger et al., WO 86/01533;Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European PatentApplication 125,023; Better et al. (1988) Science 240:1041-43; Liu etal. (1987) J Immunol 139:3521-26; Sun et al. (1987) PNAS USA 84:214-18;Nishimura et al. (1987) Canc Res 47:999-1005; Wood et al. (1985) Nature314:446-49; Shaw et al. (1988) J Natl Cancer Inst 80:1553-59; Morrison(1985) Science 229:1202-1207; Qi et al. (1986) BioTechniques 4:214;Winter, U.S. Pat. No. 5,225,539; Veroeyan et al. (1988) Science239:1534; and Beidler et al. (1988) J Immunol 141:4053-60.

Also included within the scope of the invention are antibody fragmentsand derivatives that contain at least the functional portion of theantigen binding domain of an antibody that binds specifically to anIBC-1 fragment described above. Antibody fragments that contain thebinding domain of the molecule can be generated by known techniques. Forexample, such fragments include, but are not limited to: F(ab′)₂fragments that can be produced by pepsin digestion of antibodymolecules; Fab fragments that can be generated by reducing the disulfidebridges of F(ab′)₂ fragments; and Fab fragments that can be generated bytreating antibody molecules with papain and a reducing agent. See, e.g.,National Institutes of Health, 1 Current Protocols In Immunology,Coligan et al., ed. 2.8 and 2.10 (Wiley Interscience, 1991). Antibodyfragments also include Fv (e.g., single chain Fv (scFv)) fragments,i.e., antibody products in which there are few or no constant regionamino acid residues. An ScFv fragment is a single polypeptide chain thatincludes both the heavy and light chain variable regions of the antibodyfrom which the ScFv is derived. Such fragments can be produced, forexample, as described in U.S. Pat. No. 4,642,334, which is incorporatedherein by reference in its entirety.

Methods of Diagnosis and Prognosis

This invention also features diagnostic and prognostic assays. Suchassays are based on the findings that: (a) the IBC-1 gene is expressedonly in the tumors of an aggressive subset of breast carcinomas and inthe pons, hypothalamus, and midbrain of the brain; (b) the IBC-1 gene isamplified in the same breast tumors where the IBC-1 gene is expressed;and (c) there is evidence for the existence of a cell surface IBC-1binding protein (i.e., IBC-1 receptor) in cells where IBC-1 isexpressed. Thus, detections of either (a) IBC-1 mRNA or IBC-1 in abreast cancer tissue sample or a body fluid (e.g., urine or blood) in anamount higher than in a control sample, (b) amplified genomic IBC-1 DNAin a breast cancer tissue sample, or (c) IBC-1 receptor in a breastcancer tissue sample in an amount higher than a control sample, wouldindicate that the patient has, or is likely to develop, invasive andmetastatic breast cancer. Control samples are preferably from normalsubjects, i.e., subjects without breast cancer. However, they can alsobe from patients with ductal carcinoma in situ (DCIS). Detection ofeither (a) IBC-1 mRNA or IBC-1 in a substantia nigra, pons, orhypothalamus tissue sample or in a body fluid (e.g., urine, CSF, orblood) in an amount lower than in a normal control sample; or (b) no orlower than normal amount of the IBC-1 receptor in a substantia nigra,pons, or hypothalamus tissue sample, would indicate that the patient issuffering from, or at risk for developing, a neural condition involvingdegeneration of substantia nigra, pons or hypothalamus cells. Such testscan be used on their own or, in conjunction with other procedures totest for invasive and metastatic breast cancer or degenerative neuraldiseases in appropriate subjects, e.g., human breast cancer patients, orpatients suspected of suffering from, or being at risk for developing, aneural condition involving degeneration of substantia nigra, pons orhypothalamus cells. These patients have symptoms of breast cancer,Parkinson's disease, or other neurological conditions. All humanspecimens (e.g., primary breast tumors from biopsies or surgicallyremoved tumors, and brain samples from autopsies) can be collected usingInstitutional Review Board approved protocols, snapped frozen on dryice, and stored at −80° C.

Methods of measuring mRNA levels in test cells or body fluids are knownin the art. In order to measure mRNA levels, cells in test samples canbe lysed and the levels of IBC-1 mRNA in the lysates or in RNA purifiedor semi-purified from the lysates determined by any of a variety ofmethods familiar to those in the art. Such methods include, withoutlimitation, hybridization assays using detectably labeled IBC-1-specificDNA or RNA probes and quantitative or semi-quantitative RT-PCRmethodologies using appropriate IBC-1 gene-specific oligonucleotideprimers. Alternatively, quantitative or semi-quantitative in situhybridization assays can be carried out using, for example, tissuesections or unlysed cell suspensions, and detectably (e.g.,fluorescently or enzyme-) labeled DNA or RNA probes. Additional methodsfor quantifying MRNA include the RNA protection assay (RPA), cDNA andoligonucleotide microarrays, representation difference analysis (RDA),differential display, EST sequence analysis, and SAGE.

Methods of measuring protein levels in test cells or body fluids arealso known in the art. Many such methods employ antibodies (e.g.,monoclonal or polyclonal antibodies) that bind specifically to the IBC-1protein. In such assays, the antibody itself or a secondary antibodythat binds to it can be detectably labeled. Alternatively, the antibodycan be conjugated with biotin, and detectably labeled avidin (apolypeptide that binds to biotin) can be used to detect the presence ofthe biotinylated antibody. Combinations of these approaches (including“multi-layer sandwich” assays) familiar to those in the art can be usedto enhance the sensitivity of the methodologies. Some of theseprotein-measuring assays (e.g., ELISA or Western blot) can be applied tobodily fluids or to lysates of test cells, and others (e.g.,immunohistological methods or fluorescence flow cytometry) applied tohistological sections or unlysed cell suspensions. Methods of measuringthe amount of label will be depend on the nature of the label and areknown in the art. Appropriate labels include, without limitation,radionuclides (e.g., ¹²⁵I, ¹³¹I, ³⁵S, ³H, or ³²P), enzymes (e.g.,alkaline phosphatase, horseradish peroxidase, luciferase, orβ-glactosidase), fluorescent moieties or proteins (e.g., fluorescein,rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g.,Qdot™ nanoparticles supplied by the Quantum Dot Corporation, Palo Alto,Calif.). Other applicable assays include quantitativeimmunoprecipitation or complement fixation assays.

Amplification of a gene locus can be detected by a variety of methodsknown in the art. For example, the copy number of a gene locus can bedetermined and compared by PCR amplification of genomic DNA preparedfrom a test sample and a control sample. Amplification of a gene locuscan also be identified by Southern blot analysis. Fluorescence in situhybridization (FISH) of a DNA sequence to a metaphase chromosomal spreadcan further be used to provide a precise chromosomal location and anamount of the DNA sequence present in the chromosome.

The presence of an IBC-1 receptor on the surface of a test cell can bedetermined by measuring the amount of IBC-1 bound to the cell. Methodsof measuring ligand-receptor binding in test cells are also known in theart. Many such methods involve contacting a ligand with a receptor(e.g., a receptor expressed on the surface of a cell), allowing acomplex to form between the ligand and the receptor, and detecting thebound ligand as described above. The ligand of this invention can be theIBC-1 protein itself or a receptor-binding portion of the IBC-1 protein.As used herein, “a receptor-binding portion” of the IBC-1 protein is afragment of the protein that is shorter (e.g., having 10, 20, 30, 40,50, 60, 70, 80, or 90 consecutive amino acids of IBC-1) than thefull-length protein and has at least 5% (e.g., 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 98%, 99%, 100%, or more) of the ability of thefull-length protein to bind its receptor (e.g., as measured in acompetition assay). Fragments of interest can be made by recombinant,synthetic, or proteolytic digestive methods. Such fragments can then beisolated and tested for their ability to bind an IBC-1 receptor.

Generally, the level of IBC-1 or its receptor in diseased samples willbe at least 2-fold (e.g., at least 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 10-fold, 15-fold, 20-fold, 40-fold, 60-fold, 80-fold,100-fold, 500-fold, 1,000-fold, or higher-fold) different (i.e., higherin samples from patients with invasive breast cancer, and lower insamples from patients with neural conditions involving degeneration ofsubstantia nigra, pons or hypothalamus cells) from that in the normalcounterpart samples.

Screening Assay

This invention provides methods (also referred to herein as “screeningassays”) for identifying test compounds (e.g., proteins, peptides,peptidomimetics, peptoids, antibodies, small molecules or other drugs)that block the binding of IBC-1 to its receptor. Compounds thusidentified can be used to treat conditions characterized byover-activity of IBC-1 or its receptor, e.g., invasive breast cancer.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart. Such libraries include: peptide libraries, peptoid libraries(libraries of molecules having the functionalities of peptides, but witha novel, non-peptide backbone that is resistant to enzymaticdegradation; see, e.g., Zuckernann et al. (1994) J Med Chem 37:2678-85);spatially addressable parallel solid phase or solution phase libraries;synthetic libraries obtained by deconvolution or affinity chromatographyselection; and the “one-bead one-compound” libraries. Compounds in thelast three libraries can be peptides, non-peptide oligomers or smallmolecules (Lam (1997) Anticancer Drug Des 12:145). The test compoundscan also be antibodies generated against IBC-1 fragments identified ascritical for the binding of IBC-1 to its receptor by molecular modelingor mutational analysis.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example, in: DeWitt et al. (1993) PNAS USA90:6909; Erb et al. (1994) PNAS USA 91:11422; Zuckermann et al. (1994) JMed Chem 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al.(1994) Angew Chem Int Ed Engl 33:2059; Carell et al. (1994) Angew ChemInt Ed Engl 33:2061; and Gallop et al. (1994) J Med Chem 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409),plasmids (Cull et al. (1992) PNAS USA 89:1865-1869), or phages (Scottand Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406;Cwirla et al. (1990) PNAS USA 87:6378-6382; Felici (1991) J Mol Biol222:301-310; and Ladner supra.).

To identify compounds that block the interaction between IBC-1 and itsreceptor, a reaction mixture containing IBC-1 (or a receptor-bindingportion of it) and a cell (or a cell membrane fraction or cell ghost orlipid vesicle) expressing the IBC-1 receptor is prepared underconditions and for a time sufficient to allow the two reagents to form acomplex. Cells expressing the IBC-1 receptor include certain breastcancer cells (e.g., an invasive breast cancer cell), and certain neuralcells (e.g., a substantia nigra, pons or hypothalamus cell). Such cellscan be easily identified by the techniques described herein, or bydetermining whether labeled IBC-1 binds to the cell. It has been foundthat cells that express IBC-1 also express the IBC-1 receptor.Alternatively, one can prepare such a cell by expressing a recombinantform of the receptor in a cultured cell.

The test compound can be initially included in the reaction mixture, orcan be added at a time subsequent to the addition of IBC-1 and itsreceptor. Control reaction mixtures are incubated without the testcompound. The formation of a complex between IBC-1 and its receptor isthen detected, e.g., by detecting IBC-1 bound to its receptor asdescribed above. The formation of a complex in the control reaction, butnot in the reaction mixture containing the test compound, indicates thatthe compound blocks the interaction of IBC-1 and its receptor.

Generally, a test compound whose presence reduces IBC-1 binding to itsreceptor at least 1.5 fold (e.g., at least 2-fold, 4-fold, 6-fold,10-fold, 100-fold, 1,000-fold, 10,000-fold, or 100,000-fold can beuseful as a cancer therapeutic agent. Two types of IBC-1 receptors havebeen identified: one with low affinity and one with high affinity (FIG.2C). A cell expressing a low-affinity IBC-1 receptor can be used toidentify compounds that block binding of IBC-1 to the low-affinityreceptor; a cell expressing a high-affinity IBC-1 receptor can be usedto identify compounds that block binding of IBC-1 to the high-affinityreceptor. A compound that blocks IBC-1 binding to a high-affinityreceptor is more likely to be a cancer therapeutic agent.

Methods of Treating Cancer and Degenerative Neural Diseases

This invention provides methods for treating or preventing invasive andmetastatic breast cancer. “Prevention” should mean that symptoms of thedisease (e.g., invasive and metastatic cancer) are essentially absent.Patients to be treated can be identified, for example, by determiningthe IBC-1 mRNA, IBC-1 protein, or genomic IBC-1 DNA level in a testsample prepared from a patient. If a patient has breast cancer and theIBC-1 mRNA or IBC-1 protein is present in a breast cancer tissue sampleor a body fluid at a level higher than that in a control sample, or theIBC-1 gene is amplified in a breast cancer tissue sample, the patient isa candidate for treatment with an effective amount of compound thatblocks binding of IBC-1 to its receptor.

This invention also provides methods for treating patients sufferingfrom, or at risk for developing, degenerative neural conditions. If theIBC-1 MRNA or IBC-1 protein is present in a neural tissue sample (e.g.,a substantia nigra, pons or hypothalamus tissue sample) or a body fluidfrom a patient at a level lower than normal, the patient is treated byadministering IBC-1 or its agonist to the patient such that the IBC-1 oragonist reaches the affected tissue in the brain, in an effective amountto delay, prevent, or reverse neural degeneration.

The level of IBC-1 mRNA, IBC-1 proteins, or genomic IBC-1 DNA in a testsample can be determined by methods described above, or any othermethods known in the art.

The treatment methods can be performed in vivo or ex vivo, alone or inconjunction with other drugs and/or radiotherapy.

(1) Methods of Treating or Preventing Invasive and Metastatic BreastCancer

In one in vivo approach, a therapeutic compound (e.g., a compound thatblocks the binding of IBC-1 to its receptor) itself is administered tothe subject. As used herein, a “therapeutic compound” can mean acompound the administration of which results in complete abolishment ofthe symptoms of a disease or a decrease in the severity of the symptomsof the disease. Generally, the compound will be suspended in apharmaceutically-acceptable carrier (e.g., physiological saline) andadministered orally or by intravenous (i.v.) infusion, or injected orimplanted subcutaneously, intramuscularly, intrathecally,intraperitoneally, intrarectally, intravaginally, intranasally,intragastrically, intratracheally, or intrapulmonarily. For treatment ofinvasive and metastatic breast cancer, the compound is preferablydelivered directly to tumor cells, e.g., to a tumor or a tumor bedfollowing surgical excision of the tumor, in order to kill any remainingtumor cells. For protection of breast cancer invasion and metastases,the compound can be administered (by any of the above routes) to, forexample, a patient that has not yet developed detectable invasion andmetastases but whose primary tumor was found to express IBC-1. Thedosage required depends on the choice of the route of administration;the nature of the formulation; the nature of the patient's illness; thesubject's size, weight, surface area, age, and sex; other drugs beingadministered; and the judgment of the attending physician. Suitabledosages are in the range of 0.01-100.0 mg/kg. Wide variations in theneeded dosage are to be expected in view of the variety of compoundsavailable and the different efficiencies of various routes ofadministration. For example, oral administration would be expected torequire higher dosages than administration by i.v. injection. Variationsin these dosage levels can be adjusted using standard empirical routinesfor optimization as is well understood in the art. Administrations canbe single or multiple (e.g., 2-fold, 3-fold, 4-fold, 6-fold, 8-fold,10-fold, 20-fold, 50-fold, 100-fold, 150-fold, or more fold).Encapsulation of the compound in a suitable delivery vehicle (e.g.,polymeric microparticles or implantable devices) may increase theefficiency of delivery, particularly for oral delivery.

Therapeutic compounds useful for treating or preventing metastaticbreast cancer include, but are not limited to, antagonistic fragments ofIBC-1, antibodies specific for IBC-1 (e.g., any of the IBC-1-specificantibodies disclosed herein), and/or antibodies specific for IBC-1receptor. These antibodies would of course have to be screened forantagonistic activity and lack of agonistic activity.

Alternatively, a polynucleotide containing a nucleic acid sequence thatis transcribed into an anti-sense RNA complementary to IBC-1 mRNA (thefull-length mRNA sequence or a suitable portion thereof) can bedelivered to breast cancer cells. Polynucleotides can be delivered tobreast cancer cells by, for example, the use of polymeric, biodegradablemicroparticle or microcapsule devices known in the art. Another way toachieve uptake of the nucleic acid is using liposomes, prepared bystandard methods. The vectors can be incorporated alone into thesedelivery vehicles or co-incorporated with tissue-specific ortumor-specific antibodies. Alternatively, one can prepare a molecularconjugate composed of a plasmid or other vector attached topoly-L-lysine by electrostatic or covalent forces. Poly-L-lysine bindsto a ligand that can bind to a receptor on target cells (Cristiano etal. (1995) J Mol Med 73:479). Tissue specific targeting can be achievedby the use of tissue-specific transcriptional regulatory elements (TRE)which are known in the art. Delivery of “naked DNA” (i.e., without adelivery vehicle) to an intramuscular, intradermal, or subcutaneous siteis another means to achieve in vivo expression.

The polynucleotide can include one or more sequences complementary tothe sense strand of IBC-1 DNA and a catalytic sequence known to beresponsible for mRNA cleavage (see, e.g., U.S. Pat. No. 5,093,246 andHaselhoff and Gerlach (1988) Nature 334:585-591). For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in an IBC-1 mRNA. See, e.g., U.S. Pat.Nos. 4,987,071 and 5,116,742. Alternatively, an IBC-1 mRNA can be usedto select a catalytic RNA having a specific ribonuclease activity from apool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science261:1411-1418.

In the relevant polynucleotides (e.g., expression vectors), the nucleicacid sequence encoding the anti-sense RNA is operatively linked to apromoter or enhancer-promoter combination. Enhancers provide expressionspecificity in terms of time, location, and level. Unlike a promoter, anenhancer can function when located at variable distances from thetranscription initiation site, provided a promoter is present. Anenhancer can also be located downstream of the transcription initiationsite.

Suitable expression vectors include plasmids and viral vectors such asherpes viruses, retroviruses, vaccinia viruses, attenuated vacciniaviruses, canary pox viruses, adenoviruses and adeno-associated viruses,among others.

Double-stranded interfering RNA (RNAi) homologous to IBC-1 DNA can alsobe used to reduce the production of IBC-1. See, e.g., Fire et al. (1998)Nature 391:806-811, Romano and Masino (1992) Mol. Microbiol.6:3343-3353, Cogoni et al. EMBO J. 15:3153-3163, Cogoni and Masino(1999) Nature 399:166-169, Misquitta and Paterson (1999) Proc. Natl.Acad. Sci. USA 96:1451-1456, and Kennerdell and Carthew (1998) Cell95:1017-1026.

The sense and anti-sense RNA strands can be individually constructedusing chemical synthesis and enzymatic ligation reactions usingprocedures known in the art. For example, each strand can be chemicallysynthesized using naturally occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecule or to increase the physical stability of the duplex formedbetween the sense and anti-sense strands, e.g., phosphorothioatederivatives and acridine substituted nucleotides. The sense oranti-sense strand can also be produced biologically using an expressionvector into which a target IBC-1 sequence (full-length or a fragment)has been subcloned in a sense or anti-sense orientation. The sense andanti-sense RNA strands can be annealed in vitro before delivery of thedsRNA to breast cancer cells. Alternatively, annealing can occur in vivoafter the sense and anti-sense strands are sequentially delivered to thecancer cells.

Double-stranded RNA interference can also be achieved by introducinginto breast cancer cells a polynucleotide from which sense andanti-sense RNAs can be transcribed under the direction of separatepromoters, or a single RNA molecule containing both sense and anti-sensesequences can be transcribed under the direction of a single promoter.

Polynucleotides can be administered in a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are biologicallycompatible vehicles that are suitable for administration to a human,e.g., physiological saline or liposomes. A therapeutically effectiveamount is an amount of the polynucleotide that is capable of producing amedically desirable result (e.g., decreased IBC-1 expression) in atreated patient. As is well known in the medical arts, the dosage forany one patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. Dosages will vary, but a preferred dosage foradministration of polynucleotide is from approximately 10⁶ to 10¹²copies of the polynucleotide molecule. This dose can be repeatedlyadministered, as needed. Routes of administration can be any of thoselisted above.

(2) Methods of Treating Degenerative Neural Conditions

A. In vivo Approaches

An IBC-1 or an IBC-1 agonist can be administered to a patient who has,or is likely to develop, a degenerative neural condition. Generally,IBC-1 or an IBC-1 agonist (e.g., an agonistic IBC-1 receptor antibody)will be suspended in a pharmaceutically-acceptable carrier (e.g.,physiological saline) and administered orally or by inhalation or i.v.infusion, or injected or implanted subcutaneously, intramuscularly,intrathecally, intraperitoneally, intrarectally, intravaginally,intranasally, intragastrically, intratracheally, or intrapulmonarily.For treatment of degenerative neural conditions, IBC-1, as well as thoseIBC-1 agonists small enough to cross the blood-brain barrier, does nothave to be delivered directly to neural cells, although injection of thedrug or implantation of a drug-releasing barrier device or cells in thebrain are options. The dosage required depends on the choice of theroute of administration; the nature of the formulation; the nature ofthe patient's illness; the subject's size, weight, surface area, age,and sex; other drugs being administered; and the judgment of theattending physician. Suitable dosages are in the range of 0.01-100.0mg/kg. Wide variations in the needed dosage are to be expected in viewof the variety of compounds available and the different efficiencies ofvarious routes of administration. For example, oral administration wouldbe expected to require higher dosages than administration by i.v.injection. Variations in these dosage levels can be adjusted usingstandard empirical routines for optimization as is well understood inthe art. Administrations can be single or multiple (e.g., 2-fold,3-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 50-fold, 100-fold,150-fold, or more fold). Encapsulation of the compound in a suitabledelivery vehicle (e.g., polymeric microparticles or implantable devices)may increase the efficiency of delivery, particularly for oral delivery.

B. Ex vivo Approaches

An ex vivo strategy for treating patients with degenerative neuralconditions can involve transfecting or transducing cells obtained fromthe subject with a polynucleotide encoding IBC-1, pro-IBC-1, or an IBC-1agonist. Alternatively, a cell can be transfected in vitro with a vectordesigned to insert, by homologous recombination, a new, active promoterupstream of the transcription start site of the naturally occurringendogenous IBC-1 gene in the cell's genome. Such methods, which “switchon” an otherwise largely silent gene, are well known in the art. Afterselection and expansion of a cell that expresses IBC-1 at a desiredlevel, the transfected or transduced cells are then returned to thesubject. The cells can be any of a wide range of types including,without limitation, hemopoietic cells (e.g., bone marrow cells,macrophages, monocytes, dendritic cells, T cells, or B cells),fibroblasts, epithelial cells, endothelial cells, keratinocytes, ormuscle cells. Such cells act as a source of secreted IBC-1 or an IBC-1agonist for as long as they survive in the subject.

The ex vivo methods include the steps of harvesting cells from asubject, culturing the cells, transducing them with an expressionvector, and maintaining the cells under conditions suitable forexpression of IBC-1 or an IBC-1 agonist. These methods are known in theart of molecular biology. The transduction step is accomplished by anystandard means used for ex vivo gene therapy, including calciumphosphate, lipofection, electroporation, viral infection, and biolisticgene transfer. Alternatively, liposomes or polymeric microparticles canbe used. Cells that have been successfully transduced can then beselected, for example, for expression of IBC-1 or an IBC-1 agonist. Thecells may then be injected or implanted into the patient.

The following examples are meant to illustrate, not limit, theinvention.

EXAMPLES

Materials and Methods

(1) Cell Lines and Tissue Specimens

Breast cancer cell lines were obtained from American Type CultureCollection (Manassas, Va.), or were generously provided by Drs. SteveEthier (University of Michigan), Gail Tomlinson (University of Texas),and Arthur Pardee (Dana-Farber Cancer Institute). Cells were grown inmedia recommended by the providers. Primary breast tumor samples wereobtained from Brigham and Women's Hospital, Massachusetts GeneralHospital, University Hospital Zagreb (Zagreb, Croatia), or DukeUniversity Medical Center. Immediately after removal from the patients,the samples were snap frozen on dry ice, and stored at −80° C. Brainsamples were collected from autopsies performed at Brigham and Women'sHospital or Duke University Medical Center. All human specimens werecollected using Institutional Review Board approved protocols, and allpatient identifiers were removed prior to being transported to thelaboratory.

(2) RNA Preparation, MRNA in situ Hybridization, and Northern BlotAnalysis

RNA isolation, RT-PCR and Northern blot analyses were performed asdescribed (Polyak et al. (1997) Nature 389:300-305). Human multipletissue Northern blots were purchased from Clontech (Palo Alto, Calif.).mRNA in situ hybridization using paraffin sections and digitonin-labeledriboprobes was performed following a protocol developed by St. Croix etal. (2000) Science 289:1197-1202. Frozen sections were hybridizedfollowing a protocol obtained from Dr. Qiufu Ma (Dana-Farber CancerInstitute) with minor modifications (Qian et al. (2001) Genes Dev15:2533-2545).

(3) Expression of IBC-1 in Mammalian Cells and in Bacteria

To produce recombinant IBC-1 protein in large quantities in mammaliancells, a cDNA encoding human IBC-1 without the signal sequence wasgenerated by PCR. The PCR fragment was cloned into the pSGHVO vector;this resulted in a vector encoding human growth hormone (hGH) as anamino terminal fusion partner joined to IBC-1 by a linker containing ahistidine affinity tag and a tobacco etch virus protease site (Leahy etal. (2000) Protein Expr Purif 20:500-506). For ligand binding assays,alkaline phosphatase (AP)-IBC-1 fusion proteins were generated using anAP-TAG-5 expression vector (GenHunter, Nashville, Tennessee). Mammaliancells were transfected with Fugene6™ (Roche, Indianapolis, Ind.) orLipofectamine™ (LifeTechnologies, Rockville, Md.) reagents.

For bacterial expression, the IBC-1 cDNA was PCR amplified and clonedinto pQE-30 in frame with an N-terminal hexahistidine tag. The constructwas transformed into MJ15 [pREP4] bacteria (Qiagen, Valencia, Calif.),and the recombinant IBC-1 protein was purified using denaturing bufferand Ni-NTA beads (Qiagen, Valencia, Calif.).

(4) Antibodies, Immunoblot Analyses, and in vitro Translation

A polyclonal anti-IBC-1 antibody was generated against a syntheticpeptide RQAPKPRKQRSS (SEQ ID NO:7) corresponding to amino acids 53-64 ofpro-IBC-1 (Zymed, San Francisco, Calif.). Antibodies against alkalinephosphatase and cachectic factor-1 were purchased from GenHunter(Nashville, Tenn.) and Alpha Diagnostic (San Antonio, Tex.),respectively. Immnunoblot analyses were performed as described (Krop etal. (2001) PNAS USA 98:9796-9801). Coupled in vitro transcription andtranslation reactions were performed using a PCR-generated nucleic acidencoding an IBC-1 fragment containing a C-terminal hexahistidine tag, aT7 TNT kit (Promega, Madison, Wis.) and an ³⁵S-labelled Promix™ aminoacid mixture (Amersham, Piscataway, N.J.). Proteins were purified withNiNTA beads (Qiagen, Valencia, Calif.), and resolved on 16% Tris-tricinegels (Invitrogen, Carlsbad, Calif.).

(5) Ligand Binding Assays

In vivo and in vitro ligand binding assays with primary tissues and celllines using AP-IBC-1 were performed essentially as described (Flanaganand Leder (1990) Cell 63:185-194). Briefly, frozen sections of varioushuman tissue specimens were fixed, incubated with either AP-IBC-1 fusionprotein or AP control conditioned medium, rinsed, and then incubatedwith AP substrate, forming a blue/purple precipitate. For in vitroassays, cells were incubated in suspension with conditioned mediumcontaining either AP alone or AP-IBC-1 fusion protein, rinsed, and thenassayed for the activity of bound AP. For ligand competitionexperiments, G361 cells were pre-incubated with purified recombinantHis-HID-5/psoriasin (1.93 μM) or His-IBC-1 (0.567 μM) followed byaddition of AP or AP-IBC-1.

Example 1 Identification of an IBC-1 Gene Encoding the Human Proteolysisand Cachexia Inducing Factor

Analyses of SAGE libraries derived from normal mammary epithelial cellsand in situ, invasive, and metastatic breast carcinomas identified aSAGE tag present only in libraries generated from invasive andmetastatic breast carcinomas (Porter et al. (2001) Cancer Res61:5697-5702). This tag was absent in 96 other SAGE libraries generatedfrom various human normal and cancerous tissue types (Lal et al. (1999)Cancer Res 59:5403-5407). The gene corresponding to this SAGE tag wasnamed IBC-1.

Searching the human genome sequence with a 15 base-pair sequencecontaining the IBC-1 SAGE tag and adjacent NlaIII site (CATGACGTTAAAGAC;SEQ ID NO:8), the inventors identified a genomic clone containing thistag. Using the GenScan program, it was predicted that this genomicregion encodes a gene of 4 exons with the IBC-1 SAGE tag in the lastexon following the last NlaIII site (Burge and Karlin (1997) J Mol Biol268:78-94). Based on the predicted coding sequence, primers for the most5′ and 3′ ends of the cDNA were designed, and RT-PCR analysis of mRNAderived from the breast carcinomas used for SAGE was performed. Usingthis approach, a 400 bp fragment was obtained, thus confirming that theregion encodes a transcribed gene. To confirm that the sequence of this400 bp fragment matches that of the genomic clone, the fragment wassequenced. The sequence showed that there was a small, additionaltranscribed exon not identified by the GenScan program. Therefore, thecomplete IBC-1 gene contains 5 exons, and encodes a 110 amino acidprotein containing an N-terminal signal peptide (FIGS. 1A and 1B).

The IBC-1 cDNA sequence is as follows:

(SEQ ID NO: 1)     GAAGCATGAGGTTCATGACTCTCCTCTTCCTGACAGCTCTGGCAGGAGCCCTGGTCTGTGCCTATGATCCAGAGGCCGCCTCTGCCCCAGGATCGGGGAACCCTTGCCATGAAGCATCAGCAGCTCAAAAGGAAAATGCAGGTGAAGACCCAGGGTTAGCCAGACAGGCACCAAAGCCAAGGAAGCAGAGATCCAGCCTTCTGGAAAAAGGCCTAGACGGAGCAAAAAAAGCTGTGGGGGGACTCGGAAAACTAGGAAAAGATGCAGTCGAAGATCTAGAAAGCGTGGGTAAAGGAGCCGTCCATGACGTTAAAGACGTCCTTGACTCAGTACTATAGCTGTAAGGAGAAGCTGAGAAATGATACCCAGGAGCAGCAGGCTTTACGTCTTCAGCCTAAAACCTA A

The IBC-1 cDNA (SEQ ID NO:1) is 405 nucleotides in length. The nucleicacid sequence includes an initiation codon (ATG) and a termination codon(TAG) that are underlined above. The region between and inclusive of theinitiation codon and the termination codon is a methionine-initiatedcoding sequence of 333 nucleotides including the termination codon. Thiscoding sequence is given SEQ ID NO:2. The coding sequence encodes a 110amino acid protein (SEQ ID NO:3):

(SEQ ID NO: 3)     MRFMTLLFLTALAGALVCAYDPEAASAPGSGNPCHEASAAQKENAGEDPGLARQAPKPRKQRSSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGA VHDVKDVLDSVL

The coding region of IBC-1 cDNA (SEQ ID NO:1) was found to be includedin a previously identified nucleic acid sequence encoding a humancachexia-associated protein (HCAP; Akerblom et al., U.S. Pat. No.5,834,192). However, the two sequences differ by one nucleotide withinthe 3′-untranslated region: the base at position 388 of SEQ ID NO:1(shown above in bold) is a cytosine, whereas the hcap cDNA sequence hasa thymine at the corresponding position. In addition, the predictedIBC-1 protein has very limited homology to a lacritin protein and to atranslated EST derived from the cerebral cortex (FIG. 1C). Lacritin is asecretion-enhancing and growth-promoting factor recently identified fromhuman lacrimal gland (Sanghi et al. (2001) J Mol Biol 310:127-139). TheEST expressed in the cerebral cortex encodes an uncharacterized proteincontaining a repetitive sequence ETPA found in several secretedproteins, including sialidase and neurofilamine H.

Further lower stringency searches of the Unigene and Genbank databasesusing the predicted IBC-1 amino acid sequence revealed that a portion ofIBC-1 nearly matches a 20 amino acid peptide derived from the mouse PIF(Proteolysis Inducing Factor) or cachectic factor (CF), and anoverlapping portion exactly matches the sequence of a 30 amino acidputative neural survival-promoting peptide (Cariuk et al. (1997) Br JCancer 76:606-613; and Cunningham et al. (1998) J Neurosci18:7047-7060). These polypeptides have been characterized as havingbiological and biochemical activities, but the genes encoding them havenot been identified. The neural survival-promoting peptide wasidentified from the media of mouse HN33.1 hippocampal neurons and humanY79 retinoblasts treated with hydrogen peroxide, and subsequently shownto enhance neural survival following an oxidative insult. The cachecticand proteolysis inducing factor was identified as a 24-kDa glycoproteinproduced by the cachexia-inducing MAC 16 murine colon adenocarcinoma inmice, and later shown to be present in the urine of cachectic cancerpatients (McDevitt and Tisdale (1992) Br J Cancer 66:815-820; Todorov etal. (1996) Cancer Res 56:1256-1261; and Todorov et al. (1996) Nature379:739-742). In subsequent studies, the 24 kDa cachectic factor wasshown to induce muscle protein degradation both in vivo in mice and invitro in C2C12 mouse myoblasts (Todorov et al. (1996) Cancer Res56:1256-1261; and Smith et al. (1999) Cancer Res 59:5507-5513).

The IBC-1 cDNA is predicted to encode an ˜11 kDa protein, which wasconfirmed by in vitro translation reaction. The amino acid sequence ofthe tryptic peptide obtained from the murine 24 kDa proteolysisinducing/cachectic factor (YDPEAASAPGSGNPSHEASA; SEQ ID NO:9) almostexactly matches amino acids 20-39 of the predicted IBC-1 sequence, butdoes not match to any other characterized or predicted proteins in theUnigene and Genbank databases. However, the amino acid sequence of IBC-1contains no predicted N-glycosylation sites, whereas the proteolysis andcachexia inducing protein was reported to be heavily glycosylated.

To determine if IBC-1 and the proteolysis inducing/cachectic factor areantigenically related, an immunoblot analysis of various IBC-1 fusionproteins purified from bacterial and mammalian cells was performed usinga custom made anti-IBC-1 peptide antibody and a commercially availableanti-cachectic factor peptide antibody. These analyses confirmed thatIBC-1 and the proteolysis inducing/cachectic factor are likely to beidentical or at least antigenically related, but the reason for thedifference between the reported natural (˜24 kDa) and the recombinant(˜11-13 kDa) protein sizes is unclear. One possibility is that theanti-IBC-1 antibody used in the present studies and the commercialanti-CF peptide antibodies do not recognize the glycosylated ordimerized form of the proteolysis inducing/cachectic protein. Inaddition, the inventors have not been able to express the IBC-1 proteinat detectable levels in most cell types, so it may be translated in acell type-specific manner or it may be very unstable.

Example 2 Expression Pattern of IBC-1 in Normal and Cancerous Tissues

Northern blot analyses of multiple breast tumors using an IBC-1 cDNAprobe identified a single ˜400 bp hybridizing mRNA, indicating that the400 bp cDNA fragment described above corresponds to the full-lengthtranscript. Northern blot, RT-PCR, and mRNA in situ hybridizationanalyses of normal breast organoids (freshly isolated mammary ducts),primary breast carcinomas, and breast cancer cell lines demonstratedthat IBC-1 is not expressed in normal mammary epithelium nor in themajority of breast cancer cell lines and tumors. Interestingly, all thebreast tumors (6 out of 55 total) that showed high IBC-1 expressionlevels were poorly differentiated (grade III) and stronglyerbB2-expressing tumors, and five out of the six IBC-1 positive tumorshad multiple metastatic lymph nodes. However, due to the relativelysmall sample size, among these tumor characteristics, only theoverexpression of erbB2 showed a nearly significant (Fisher exact testP=0.06) association with IBC-1 expression. These data indicate thatIBC-1 expression is not a common event in breast carcinomas, but definesa particularly aggressive tumor phenotype.

Analyses of 100 SAGE libraries derived from multiple normal andcancerous human tissues and cell lines suggested that IBC-1 is expressedonly in a subset of breast carcinomas. To further investigate IBC-1expression, the IBC-1 cDNA was hybridized against a tissue expressionarray panel containing mRNA from 76 normal human adult and fetal tissuetypes. IBC-1 was found to be expressed only in two regions of the brain:in the pons, and at a lower level, in the paracentral gyrus of thecerebral cortex. This restricted expression pattern suggests that IBC-1would be useful as a breast cancer diagnostic or prognostic marker. Inaddition, these observations imply that IBC-1-expressing tumors may haveacquired a neuronal phenotype. In order to test the latter hypothesis,the expression of several neural markers (e.g., chromogranin A,synaptogenin, neuronal enolase) in the breast tumor samples was analyzedby Northern blot or SAGE analysis, but found no correlation between theexpression of these genes and that of IBC-1.

The SAGE libraries from which IBC-1 was identified were generated fromunpurified invasive and metastatic breast carcinomas containing stromalfibroblasts, lymphocytes, endothelial cells, and other cell types. Tocharacterize the expression of IBC-1 at the cellular level, mRNA in situhybridization was performed on sections of two tumors known to expressIBC-1 based on Northern blot analysis and ten additional tumors thatwere similar to them based on expression profiling and clusteringanalysis. Four out of the latter ten tumors contained some IBC-1positive tumor cells, further suggesting that IBC-1 expression defines abiologically relevant subset of breast carcinomas. Intense red or black(depending on the MRNA in situ hybridization method used) staining inthe anti-sense slide demonstrates that IBC-1 is expressed in tumorcells, but not in stromal fibroblasts, endothelial cells, or lymphoidcells. No signal was observed in adjacent normal mammary epithelialcells. Interestingly, in some samples, all tumor cells were stronglyIBC-1 positive, while in others, only a subset of the tumor cells showedhigh IBC-1 expression, indicating that IBC-1 is useful in identifyingintra-tumoral clonal heterogeneity.

To analyze the expression of IBC-1 at the protein level, a rabbitpolyclonal anti-IBC-1 antibody was generated using a synthetic IBC-1peptide as an immunogen and affinity-purified. To determine whether theexpression of IBC-1 correlates with histo-pathologic or clinicalcharacteristics of breast tumors, an immunohistochemical analysis of 722breast tumors collected from 8 different cohorts was performed using thepolyclonal anti-IBC-1 antibody. Overall, only 6-11% (depending on thepatient cohort) of these tumors were IBC-1 positive. Statisticalanalysis of the immunohistochemistry data indicated that the expressionof IBC-1 was not significantly different between (a) in situ and primaryinvasive tumors and (b) distant metastases, although the number of insitu tumors and distant metastases were relatively low and only 1 ductalcarcinoma in situ (DCIS) was found to be IBC-1 positive. Similarly, noassociation was found between (a) IBC-1 expression and (b) estrogen andprogesterone receptor status, tumor size, the number of positive lymphnodes and the age of the patient. However, the expression of IBC-1correlated positively with erbB2 expression in a subset of the tumors.importantly, based on a logistic regression model, IBC-1 positiveprimary tumors were more likely to be stage 2 and 3 than stage 1 (LRtest, p-value=0.007), suggesting a role for IBC-1 in tumor progression.Finally, patients with IBC-1 positive tumors were somewhat more likelyto have a shorter disease free survival (calculated as time untildistant metastasis) and overall survival than patients with IBC-1negative tumors. Although this association did not reach statisticalsignificance (p-value=0.8 for overall survival and p-value=0.43 fordisease free survival) due to the relatively low number of IBC-1positive tumors with clinical data, it suggests that IBC-1 expressioncan be an independent indicator of poor prognosis.

Example 3 Identification of Putative Cell Surface IBC-1 BindingProtein(s)

Both cachectic factor and the neural survival peptide were identified assecreted proteins. Consistent with that, the IBC-1 cDNA encodes a 110amino acid protein with a predicted 19 amino acid secretory signalpeptide. These data indicate that IBC-1 is likely to execute itsfunction through binding to a cell surface receptor. To determine ifthere is an IBC-1-binding cell surface protein(s), an alkalinephosphatase-IBC-1 (AP-IBC-1) fusion protein to be used as a ligand inreceptor binding assays was generated (Flanagan and Leder (1990) Cell63:185-194; FIG. 2A). Conditioned medium of AP-IBC-1 or control APexpressing cells was used as an affinity reagent, much like an antibody,to stain normal and cancerous mammary tissue sections. Intense purplestaining indicates the presence of an IBC-1-binding protein in invasivebreast carcinoma with high endogenous IBC-1 expression, but not innormal mammary epithelial and stromal cells. Interestingly, tumors thatexpress IBC-1 demonstrated much more intense staining than did tumorswith low or undetectable endogenous IBC-1 expression. These resultsindicate the presence of a cell surface IBC-1-binding protein incancerous but not normal mammary epithelial cells in vivo, and indicatean autocrine mechanism of IBC-1 action.

Because IBC-1 is expressed in neurons of the pons, hypothalamus andmidbrain, and because of IBC-1's role in cachexia, these tissues weretested for their ability to bind IBC-1. Surprisingly, weak IBC-1 bindingto almost all neurons was seen. The most intense alkaline phosphatasestaining (i.e., the strongest IBC-1 binding) was detected in neurons ofthe locus ceruleus, nucleus raphe pontis, substantia nigra, and thelateral hypothalamic nuclei, also known as the lateral hypothalamic areaor zone.

To further test the binding characteristics of AP-IBC-1, in vitro ligandbinding assays were performed on various cell lines (FIG. 2B). Low levelAP-IBC-1 binding was detected in all cell lines tested, and strongerbinding was observed in human 21NT breast cancer, G361 melanoma, mouseC2C12 myoblast, and CATH.a catecholaminergic neurons. The IBC-1 bindingactivity was completely abolished by pretreatment of cells with trypsin,indicating that the IBC-1 binding activity is likely due to the presenceof a cell surface protein. Addition of purified recombinant IBC-1, butnot HID-5/psoriasin, significantly reduced AP-IBC-1 binding to G361cells, indicating that the binding is IBC-1-specific. 21iNT and G361cells express IBC-1; IBC-1 was shown to induce proteolysis in C2C12(Smith et al. (1999) Cancer Res 59:5507-5513; and Todorov et al. (1999)Br J Cancer 80:1734-1737). CATH.a cells were derived from a brain tumorof transgenic mice expressing the SV40 T antigen under the control ofthe rat tyrosine hydroxylase gene promoter (Suri et al. (1993) JNeurosci 13:1280-1291). CATH.a cells have catecholaminergic neuronalphenotype, and are highly sensitive to apoptosis induced by dopamine andhydrogen-peroxide (Masserano et al.(1996) Mol Pharmacol 50:1309-1315).Although the amount of bound AP-IBC-1 may be influenced by cell size,the difference in the size of these cells is unlikely to accountentirely for the observed differences in AP-IBC-1 binding. Therefore,cells that respond to IBC-1 are likely to express an increased number ofor higher affinity IBC-1 binding proteins.

To further characterize the AP-IBC-1-putative IBC-1 receptorinteraction, more detailed binding assays were performed on G361melanoma cells (FIG. 2C). Scatchard plot analysis shows two bindingslopes: one with a moderately high affinity (Kd=37.5 nM, 1.7×10⁴ bindingsites/cell) and another with much lower affinity (Kd=360 nM, 3.5×10⁵binding sites/cell).

Example 4 Amplification of IBC-1 Gene in Breast Tumors

The IBC-1 gene is localized to chromosome 12q13, an area previouslyimplicated in various malignancies. Intriguingly, based on our SAGEdata, two genes nearest to IBC-1 (LACRT and PPPlRlA) were also highlyand specifically expressed in the same tumor samples that highlyexpressed IBC-1. This led us to hypothesize that the overexpression ofIBC-1 in these breast tumors may be due to genetic amplification. Inorder to test this hypothesis, a bacterial artificial chromosome (BAC)containing the IBC-1 gene was isolated and used for FISH (fluorescent insitu hybridization) analysis of normal breast tissue and four breasttumors that overexpressed IBC-1 (as determined by SAGE or Northernblot/mRNA in situ hybridization). The analysis showed that there is amoderate- to high-level gain of the IBC-1 genomic locus in tumors thatoverexpress IBC-1. Since several known oncogenes, including CDK4, SAS,GLI, and MDM2, are also localized to chromosome 12q13-15, FISH analysesusing BACs corresponding to these genes was carried out to determine ifthey are co-amplified with IBC-1. MDM2 and GLI1 were not amplified inthese tumors, while in a subset of the cells, both CDK4 and IBC-1 wereamplified. However, in tumors that overexpressed IBC-1, there was noevidence of CDK4 overexpression. These data show that a subset ofinvasive breast tumors overexpress and amplify IBC-1.

Example 5 Polyclonal IBC-specific Antibodies

As a custom antibody service ordered by the inventors, ZymedLaboratories, Inc., South San Francisco, Calif., produced twoIBC-1-specific rabbit polyclonal antibody preparations by immunizing(using standard procedures) rabbits with three different syntheticpeptides. One group of rabbits was immunized with a N-terminal IBC-1synthetic peptide fragment consisting of amino acids 45-64 of pro-IBC-1(AGEDPGLARQAPKPRKQRSS; SEQ ID NO:12), a second group of rabbits wasimmunized with a N-terminal IBC-1 synthetic peptide fragment consistingof amino acids 53-64 of pro-IBC-1 (RQAPKPRKQRSS; SEQ ID NO:7), and athird group of rabbits was immunized with a C-terminal IBC-1 syntheticpeptide fragment consisting of amino acids 86-103 of pro-IBC-1(DAVEDLESVGKGAVHDVK; SEQ ID NO:13). Antibodies specific for theC-terminus of IBC-1 are especially useful in that the C-terminus ofIBC-1 is more stable than the N-terminus and thus antibodies specificfor the C-terminus provide a particularly sensitive means for testingfor the expression of, or the presence of, IBC-1 protein in, forexample, test cells (e.g., breast epithelial cells), tissues, or bodilyfluids (e.g., blood, urine, or sweat) of interest. The inventors found,by western blot analysis, that the three polyclonal antibodies producedby immunizing rabbits with the above-described three IBC-1 syntheticpeptide fragments bound to: recombinant IBC-1 produced in bacteria;IBC-1 expressed in breast cancer cells; and IBC-1 present in sweat (datanot shown).

OTHER EMBODIMENTS

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A purified monoclonal antibody, or antigen-binding fragment thereof,that specifically binds to an epitope within the sequence of SEQ ID NO:6(amino acids 43-64 of pro-IBC-1).
 2. A purified monoclonal antibody, orantigen-binding fragment thereof, that specifically binds to an epitopewithin the sequence of SEQ ID NO:12 (amino acids 45-64 of pro-IBC-1). 3.A purified monoclonal antibody, or antigen-binding fragment thereof,that specifically binds to an epitope within the sequence of SEQ ID NO:7(amino acids 53-64 of pro-IBC-1).
 4. A purified monoclonal antibody, orantigen-binding fragment thereof, that specifically binds to an epitopewithin the sequence of SEQ ID NO:13 (amino acids 86-103 of pro-IBC-1).5. The monoclonal antibody, or antigen-binding fragment thereof, ofclaim 1, wherein the antibody is a chimerie antibody.
 6. The monoclonalantibody, or antigen-binding fragment thereof, of claim 1, wherein theantibody is a humanized antibody.
 7. The monoclonal antibody, orantigen-binding fragment thereof, of claim 1, wherein theantigen-binding fragment is a single chain Fv fragment.
 8. Themonoclonal antibody, or antigen-binding fragment thereof, of claim 4,wherein the antibody is a chimeric antibody.
 9. The monoclonal antibody,or antigen-binding fragment thereof, of claim 4, wherein the antibody isa humanized antibody.
 10. The monoclonal antibody, or antigen-bindingfragment thereof, of claim 4, wherein the antigen-binding fragment is asingle chain Fv fragment.
 11. The monoclonal antibody, orantigen-binding fragment thereof, of claim 2, wherein the antibody is achimeric antibody.
 12. The monoclonal antibody, or antigen-bindingfragment thereof, of claim 2, wherein the antibody is a humanizedantibody.
 13. The monoclonal antibody, or antigen-binding fragmentthereof, of claim 2, wherein the antigen-binding fragment is a singlechain Fv fragment.
 14. The monoclonal antibody, or antigen-bindingfragment thereof, of claim 3, wherein the antibody is a chimericantibody.
 15. The monoclonal antibody, or antigen-binding fragmentthereof, of claim 3, wherein the antibody is a humanized antibody. 16.The monoclonal antibody, or antigen-binding fragment thereof, of claim3, wherein the antigen-binding fragment is a single chain Fv fragment.